Sensing of luminal contents by the gastrointestinal (GI) mucosa plays a critical role in the regulation of digestive functions and protection from harmful substances. The recent discovery that bitter taste receptors (T2Rs) and the G-protein subunits, ??gustducin and ??transducin, which mediate gustatory signals in the oral cavity, are also expressed in the GI mucosa suggests that these signaling molecules participate in the functional detection of harmful substances in the lumen and possibly initiate a protective response including cessation of food intake. This application will test the hypothesis that activation of bitter chemosensory receptors in the GI mucosa induces release of signaling molecules by epithelial cells that in turn activate neuronal pathways to modulate GI function and food intake. This hypothesis will be tested by the following specific aims. Specific Aim 1 will determine 1a) the effects and site of action along the gut of intraluminal T2R agonists on vagal afferent pathways using c-fos as marker of neuronal activity, and the role of CCK and PYY, peptides that affect GI function and feeding behavior, acting at CCK1 and Y2 receptors on vagal afferents, 1b) the effects of T2R agonists on vagal pathways using electrophysiological recording of vagal afferents innervating the stomach and duodenum, and 1c) whether afferent neuronal activation induced by T2Rs agonists is mediated by ??gustducin and ??transducin. Specific Aim 2 will establish the functional significance of the stimulation of gastrointestinal T2Rs and their regulation by feeding by determining 2a) the changes in gastric motor function, intestinal secretion, food intake and aversion behavior in response to T2R subtype agonists, 2b) whether these effects are mediated by ?? gustducin and ??transducin and whether they involve CCK and PYY acting at CCK1 and Y2 receptors, 2c) the effect of fasting and feeding, and of bitter stimulation on the expression of ??gustducin, ??transducin and selected T2Rs, and 2d) the signal transduction pathways (intracellular Ca2+ and ERK) initiated by T2R agonists in enteroendocrine STC-1 cells in vitro, and whether intraluminal T2R agonists activate endocrine cells in situ using immunohistochemistry for phosphorylated calmodulin dependent kinase 2 (CAMK2) as a marker for intracellular Ca2+ elevation. The long term goal is to develop an understanding of the mechanisms regulating luminal chemosensing. This is of importance since molecular sensing of gut luminal contents regulates motility, release of signaling molecules and homeostasis maintenance, and it is also responsible for the detection of ingested harmful drugs and toxins that could initiate response critical for survival. PUBLIC HEALTH RELEVANCE: Sensing of luminal contents by the gastrointestinal (GI) mucosa plays a critical role in the regulation of digestive functions and in the protection from harmful substances. The current application will focus on the pathways activated by bitter tastants with different affinity for different taste receptors and the effects of these agonists on GI function and feeding behavior. The long term goal is to develop an understanding of the mechanisms regulating luminal chemosensing, an important physiological process that controls GI functional responses that impact on feeding behavior and protection from harmful drugs and toxins.